Microprojectile delivery system and particulate product

ABSTRACT

The invention relates to a particulate product comprising at least one microprojectile; characterized in that the or at least one of the microprojectiles comprises silicon. The invention also relates to devices and components used in the microprojectile implantation of the particulate product to a target of cells or target tissue.

BACKGROUND OF THE INVENTION

The present invention relates to new products that may be implanted intocells or a target tissue. The invention also relates to components anddevices that may be used in the delivery of said products to cells andtissues. In a further aspect, the invention relates to methods offabricating said products, components, and devices. In a yet furtheraspect the invention relates to a new particulate product.

It is known that gold particles coated with DNA may be used to transferDNA into cells. This is achieved by accelerating the particles towards atarget of cells. The particles then pass through the cell walls and/ormembranes carrying the DNA into the cell. Compressed gas such as heliumis commonly used to bring about this acceleration.

A similar technique has also been developed to inject particles throughthe skin of a patient. Again the particles are accelerated by compressedgas and pass through the skin into the body of the patient. Theparticles can be used to inject drugs, DNA, or vaccines to the bloodstream or tissues of humans or animals.

The implantation of particles into tissue or cells in this way is knownas microprojectile implantation, and involves acceleration of a particleto a velocity that allows it to penetrate a cell wall and/or membrane orto penetrate tissue. Microprojectile implantation differs from otherforms of implantation since it is the momentum of the particle thatcauses the breach in the cell wall or tissue, as opposed to an implementsuch as a needle or surgeon's knife. A further factor that affectsimplantation depth and degree of tissue damage is the shape of themicroprojectile particle.

Microprojectile implantation has several advantages over other forms ofimplantation. The technique makes it easier for human patients to selfadminister an active substance, eliminating the use of needles. Theactive substance can be used in dry form, potentially increasingstability of many active substances. The procedure is significantly lesspainful than needle delivery and is hence particularly favoured forpaediatric use. Finally since the active substance can be delivered inparticulate form the release of the substance may, in certaincircumstances, be better controlled.

A microprojectile is a particle having a composition, size, shape, andmass such that it is suitable for microprojectile implanation into atarget tissue or cell, or into the blood stream of a patient. If themicroprojectile is being administered to tissue (eg skin), the velocityand momentum must be set to achieve the correct level of penetration inorder to achieve the desired physiological effect. Microprojectiles aretypically used in association with an active substance, such as a drugor biological material. The properties that make the particle suitablefor microprojectile implantation will depend upon the active substanceto be delivered to the target, upon the technique used to deliver theparticles, and upon the target tissue or cell. For example if amicroprojectile is to be introduced into a cell, then its constitutionand velocity must allow it to penetrate the cell wall and/or cellmembranes without destroying the cell. Typically the particle must beapproximately one tenth the size of the cell to be implanted. If, on theother hand, it is for extracellular drug delivery, its size issignificantly larger, and often in the 10 to 100 micron range.

The active substance may be coated onto the microprojectile, for exampleDNA may be precipitated onto the surface of gold particles. In the caseof a drug to be implanted in a patient, the microprojectile may simplyconsist of an excipient combined with the drug. A relatively low densitymaterial such as ice may be used as a carrier material for the activesubstance: the substance may be dissolved or otherwise combined withwater; the solution/suspension is then nebulised and the resultingdroplets frozen. The frozen droplets can then be implanted into thecells where the ice melts releasing the substance.

A number of devices may be used to deliver microprojectiles to thetarget cells or tissue. Such devices (delivery devices) typicallycomprise a gas source and a component (a carrier component) forretaining the microprojectiles prior to delivery. The gas source isoften a small pressurised helium cylinder and can be activated bypuncturing the cylinder to release a flow of helium. The device, oftentermed a gene gun if the material to be delivered is genetic, may bearranged so that the flow of helium causes the microprojectiles to beaccelerated towards the target. For example the carrier component maycomprise a disc upon which the microprojectiles are adhered, the flow ofgas causing them to be dislodged from the disc. The carrier componentand gas source are usually designed to facilitate their replacement sothat the microprojectile delivery device may be used many times.

It is known that products may be implanted in human or animal patientsby techniques other than microprojectile implantation. For example animplant may be introduced surgically or by injection though a needle.Both these techniques are referred to in PCT/GB99/01185, which describesthe use of porous and polycrystalline silicon implants. There areseveral types of porous and polycrystalline silicon including:biocompatible silicon, bioactive silicon, and resorbable silicon. Thefabrication and properties of these three types of silicon are referredto in PCT/GB96/01863.

There are a number of problems associated with existingmicroprojectiles. Many microprojectiles currently used are only able tocarry a small amount of active substance in relation to their size.Prior art microprojectiles are typically solid, so that the activesubstance is confined to the surface of the microprojectile. The surfacelocation of the active substance means that it is exposed to forcesduring passage of the microprojectile into the target, and is thereforevulnerable to damage. Where the active substance comprises large organicmolecules such as DNA, then passage of the microprojectiles through theskin of a patient may cause the DNA molecules to fragment. The immuneresponse of a patient may also cause deactivation of the substance as aresult of its surface location.

Prior art microprojectiles, which have sufficient mechanical strength towithstand the forces of implantation, are typically fabricated frommaterials that are insoluble in biological environments. This can hinderthe release of the active substance into the cell or tissue. For exampleDNA present on the surface of gold particles is immobilised by the gold,hindering transfection of the DNA. This immobilisation means that theDNA may be degraded before it can be intercalated into the nucleus ofthe implanted cell. Though in some cases it may be advantageous for theDNA to remain on the gold in an active form. Another material commonlyused in the fabrication of microprojectiles is tungsten. Tungsten isinexpensive relative to gold, but it suffers from several disadvantages.It is difficult to fabricate tungsten microprojectiles having as uniformsize distribution. Tungsten is also potentially toxic; and finally isalso known to catalytically degrade DNA bound to its surface.

The factors that affect whether a material is suitable for use inmicroprojectile implantation are therefore complex. Properties such asdensity, toxicity, mechanical strength, internal structure, surfaceproperties, and solubility in a variety of environments, may all affectthe performance of the material.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide products that bettersatisfy the requirements associated with microprojectile delivery of anactive substance to cells or tissue. It is a further object of thepresent invention to provide components and devices that also bettersatisfy the requirements associated with microprojectile delivery of anactive substance to a cells or tissue. It is a yet further object of theinvention to provide methods of fabricating said products, components,and devices.

According to a first aspect the invention provides a particulate productcomprising at least one silicon particle.

The or each silicon particle comprises silicon.

The or at least one of the silicon particles may comprise one or moreof: porous silicon, polycrystalline silicon, resorbable silicon,bioactive silicon, bulk crystalline silicon, biocompatible silicon, andamorphous silicon.

If the particulate product comprises more than one silicon particle,then the silicon particles from which the product is formed need not allcomprise the same type of silicon. For example some of the particles maycomprise porous silicon and others may comprise bulk crystallinesilicon.

Advantageously the or at least one of the silcon particles is amicroprojectile.

For the avoidance of doubt a microprojectile is a particle having acomposition, size, shape, and mass such that it is suitable formicroprojectile implanation into a target tissue or cell, or into theblood stream of a patient.

Preferably the microprojectile has a composition, size, shape and masssuch that it is suitable for microprojectile implantation into a humanor animal.

Advantageously the or at least one of the microprojectiles has anelongated shape. More advantageously the or at least one of themicroprojectiles has a pointed tip. Yet more advantageously the or atleast one of the microprojectiles comprises a microneedle.

The or at least one of the microprojectiles may comprise a microbarband/or a microdart.

Preferably the or at least one of the microprojectiles comprises porousand/or polycrystalline silicon.

Advantageously the or at least one of the microprojectiles has acomposition, size, shape, and mass such that they are suitable for usein one or more of the devices disclosed in U.S. Pat. Nos. 5,204,253;5,219,746; 5,506,125; 5,584,807; 5,865,796; 5,877,023; 5,919,159;6,004,287; and 6,013,050, which are hereby incorporated by reference.

The particulate product may comprise a multiplicity of microprojectiles,the microprojectiles forming a powder. The powder may have asubstantially uniform particle size distribution.

A number of advantages are associated with the use of silicon,particularly porous and/or polycrystalline silicon, in the fabricationof microprojectiles.

Porous and polycrystalline materials, if they have suitablenanostructure, exhibit visible-near infrared fluorescence. Thefluorescence of the porous and polycrystalline silicon may be of valuein monitoring drug concentrations in the blood of a patient, as well asthe presence and quality of the implanted microprojectile. This is ofparticular value silicon devices comprising resorbable silicon. Wherethe blood of a patient contains microprojectiles with which a drug hasbeen combined, analysis of a blood sample containing themicroprojectiles could yield information about the drug andmicroprojectile concentration. The fluorescence allows themicroprojectiles to be identified and their numbers to be determinedwith relative ease.

The fabrication of microprojectiles from silicon allows the use ofsilicon processing technologies. These silicon processing techniques, inturn, open the way for exquisite control over microprojectile shape andsize, coupled with high yields and high purity products. Better controlover not only the size, but also the shape of an assembly ofmicroprojectiles will result in better control of the depth ofpenetration of tissue or their incorporation within target cells. Theuse of porous silicon microprojectiles is also advantageaous since thepresence of pores allows a greater dose, and flexibility of loading of,active substance to be delivered for a given microprojectile size.

The or at least one of the microprojectiles may further comprise a highdensity material having a density greater than that of bulk crystallinesilicon. The high density material may comprise one or more of: gold,tungsten, platinum, iron, nickel, molybdenum, silver, palladium, erbium,iridium, rhenium, and cobalt. The microprojectile may comprise asilicide. The silicon, from which the microprojectile is formed, may belocated at the surface of the high density material.

The use of a material having a density greater than that of bulkcrystalline silicon may be of particular value in intercellularmicroprojectile delivery.

The or at least one of the microprojectiles may comprise bulkcrystalline silicon.

The or at least one of the microprojectiles may have a mass in the range0.001 ng to 5 ng. The or at least one of the microprojectiles may have amass in the range 1 ng to 1 μg. The or at least one of themicroprojectiles may have a mass in the range 1 ng to 5 ng.

The 1 ng to 1 μg mass range may be of particular value in the deliveryvaccines to cells. The 0.001 ng to 1 ng mass range may be of particularvalue in drug delivery to target tissues.

Advantageously the or at least one of the silicon particles furthercomprises an active substance.

The active substance may comprise one or more of: a pharmaceuticalmaterial, a biological material, a genetic material, a radioactivematerial, an antibacterial agent, and luminescent agent.

The active substance may comprise one or more of: insulin, lidocaine,anaesthetic, alprostadil, calcitonin, DNA, RNA, peptide, cytokine,hormone, antibody, cytotoxic agent, adjuvant, steroid, and protein.

The active substance may comprise one or more of: GnRH, Goserilin,Leuprordin Acetate, Triptordin, Buserelin, a GnRH agonist, a GnRHsuperagonist, a GnRH antagonist, a GnRH homologue, a GnRH analogue, anda GnRH mimic.

For the purposes of this specification the term active substance meansany substance to be transferred into a target cell or tissue or into apatient.

Advantageously the active substance comprises DNA or RNA.

The active substance may be disposed, at least partly, in the interiorof the or at least one of the silicon particles. The or at least one ofthe silicon particles may comprise porous silicon and the activesubstance may be disposed, at least partly, in the pores of the poroussilicon. Alternatively the or at least one of the silicon particles maycomprise a cavity that is bounded, at least partly, by the silicon. Theactive substance may be disposed in said cavity.

If the silicon particle comprises an active substance that is disposedin a cavity at least partly bounded by the silicon, or that is disposedin the pores of porous silicon; then the active substance will beprotected from the effect of implantation into the cells or tissue. Forexample if the active substance comprises DNA, then the DNA will beprotected from shearing forces as it passes through the tissue or cellwalls.

Preferably the or at least one of the microprojectiles comprisesresorbable silicon.

Advantageously the silicon comprises derivatised silicon. Moreadvantageously the silicon comprises derivatised porous and/orpolycrystalline silicon. Yet more advantageously the derivatised siliconcomprises one or both of: Si—C bonding, and Si—O—C bonding.

As is well known in the art, the term “derivatised porous and/orpolycrystalline silicon” means porous and/or polycrystalline siliconthat has been derivatised predominantly or exclusively at the surface ofthe silicon.

By selecting appropriate derivatisation, the surface functionality ofthe microprojectile may be tailored to meet the requirements of theactive substance.

The use of resorbable silicon is of value since resorbable silicon isknown to dissolve or corrode in biological environments. The use of anactive substance associated with resorbable silicon therefore opens theway for the controlled release of the active substance as a result ofthe corrosion/dissolution of the resorbable silicon. If the resorbablesilicon is porous then the active substance may be disposed in the poresof the porous silicon; corrosion of the silicon may then release thesubstance from the pores. If the microprojectile comprises a cavity, inwhich an active substance is disposed and which is bounded by resorbablesilicon, then corrosion of the silicon may also result in release of thesubstance.

The use of resorbable silicon microprojectiles potentially allows thedelivery of large quantities of an active substance, relative to priorart microprojectiles. For example gold microprojectiles are only able todeliver active substances from the surface of the microprojectile. Theuse of resorbable silicon allows delivery of the whole active substancepayload, throughout the volume of the microprojectile.

Advantageously the or at least one of the silicon particles comprisesporous silicon having a porosity between 1% and 90%. More advantageouslythe porous silicon has a porosity between 10% and 80%.

Preferably the or at least one of the silicon particles may have a sizein the range 100 nm to 500 μm. More preferably the or at least one ofthe silicon particles has a size in the range 100 nm to 250 μm.

The or at least one of the silicon particles may have a size in therange 10 μm to 100 μm. The or at least one of the silicon particles mayhave a size in the range 10 μm to 70 μm. The or at least one of thesilicon particles may have a size in the range 1 μm to 15 μm.

The size range 10 μm to 100 μm may be of particular value forextracellular drug microprojectile delivery. The size range 1 μm to 15μm may be of particular value for intracellular microprojectiledelivery.

Advantageously the particulate product comprises at least fivesubstantially single sized silicon particles, each single sized siliconparticle having a volume that is substantially identical to the volumeof the other single sized particles. More advantageously the particulateproduct comprises at least ten substantially single sized siliconparticles, each single sized silicon particle having a volume that issubstantially identical to the volume of the other single sizedparticles. Yet more advantageously the particulate product comprises atleast twenty substantially single sized silicon particles, each singlesized silicon particle having a volume that is substantially identicalto the volume of the other single sized particles.

Preferably the particulate product comprises a multiplicity of singleshaped silicon particles, each single shaped silicon particle having thesubstantially same shape as the other single shaped silicon particles.More advantageously each single shaped silicon particle has thesubstantially the same volume as the other single shaped siliconparticles. Yet more advantageously each single shaped silicon particleis substantially symmetric.

The total mass of the single shaped silicon particles, from which theparticulate product is at least partly formed, may be greater than 10%of the total mass of the particulate product. The total mass of thesingle shaped silicon particles, from which the particulate product isat least partly formed, may be greater than 50% of the total mass of theparticulate product.

The particulate product may comprise a multiplicity of silicon particlesand at least some of said silicon particles may be monodispersed.

Advantageously the or at least one of the silicon particles issubstantially symmetric. More advantageously the particualte productcomprises a multiplicity of substantially symmetric silicon particles,each substantially symmetric silicon particle being substantiallysymmetric.

The or at least one of the silicon particles may be substantially cubic.The or at least one of the silicon particles may be substantiallyspherical.

For the purposes of this specification the term “symmetric”, when usedto describe an object, means that the object comprises at least oneplane of symmetry and/or at least one axis of symmetry.

According to a second aspect the invention provides a carrier component,for use in microprojectile implantation, comprising a carrier body andat least one microprojectile, the carrier body having a shape and beingarranged such that the carrier body retains the or at least one of themicroprojectiles, characterised in that the or at least one of themicroprojectiles comprises silicon.

Carrier components (and hence the carrier body) are usually used inmicroprojectile delivery devices. They are designed to facilitateremoval and replacement of the component in the device.

The carrier body may have a shape and be arranged such that it forms acartridge, the or at least one of the microprojectiles being disposedwithin the cartridge. The carrier body may comprise a carrier wall, theor at least one of the microprojectiles being adhered to, or integralwith, said carrier wall.

Advantageously the microprojectile comprises porous and/orpolycrystalline silicon.

According to a third aspect, the invention provides a delivery devicecomprising at least one microprojectile and an activatable gas source;the gas source being arranged such that, when activated, it causes gasto impart kinetic energy to the or at least one of the microprojectiles;characterised in that the or at least one of the microprojectilescomprises silicon.

The gas may impart kinetic energy to the microprojectiles by directimpact of the gas with the microprojectiles. Alternatively themicroprojectiles may be adhered to one side of a disc; in which case themicroprojectiles may be accelerated by impact of the gas with the discsurface opposite to that on which the particles are adhered.

When the device is arranged appropriately, impact of the gas with themicroprojectiles (or a body to which the microprojectiles are adhered)causes them to accelerate towards the target cells or tissue.

The gas source may comprise a reservoir, containing gas held underpressure and having a reservoir wall that encloses the gas. The gassource may be activated by rupturing the wall, thereby causing the gasto flow from the interior to the exterior of the reservoir.

The microprojectile delivery device need not comprise a reservoir ofgas. The gas source may, for example, simply comprise a gas conduitattached by a tube to a cylinder; the cylinder being separate from thedevice. In this case the gas source may be activated by opening a valvebetween the tubing and the conduit.

The gas source may comprise an explosive, such as gunpowder; the gassource being activated by ignition of the explosive.

The microprojectile delivery device may further comprise a carrier bodythe carrier body having a shape and being arranged such that the carrierbody retains the or at least one of the microprojectiles.

Preferably the microprojectile comprises porous and/or polycrystallinesilicon.

According to a fourth aspect, the invention provides a method offabricating a particulate product comprising the steps: (a) taking asample of silicon and (b) forming at least one silicon product particlefrom the sample of silicon.

The silicon sample comprises silicon and the or each of the siliconproduct particles comprises silicon.

The sample of silicon may comprise one or more of: porous silicon,polycrystalline silicon, resorbable silicon, bioactive silicon, bulkcrystalline silicon, biocompatible silicon, and amorphous silicon.

The or at least one of the silicon product particles may comprise one ormore of: porous silicon, polycrystalline silicon, resorbable silicon,bioactive silicon, bulk crystalline silicon, biocompatible silicon, andamorphous silicon.

Preferably step (b) is performed in such a manner that at least one ofthe silicon product particles is a microprojectile.

Advantageously step (b) is performed in such a manner that at least oneof the silicon product particles is symmetric. Yet more advantageouslystep (b) is performed in such a manner that a multiplicity of symmetricsilicon product particles.

Step (b) may be performed in such a manner that at least five singlesized silicon product particles are formed, each single sized siliconparticle having a volume that is substantially identical to the othersingle sized product silicon particles.

Step (b) may be performed in such a manner that a multiplicity of singleshaped product silicon particles are formed, each single shaped siliconproduct particle having a shape that is substantially identical to theother single shaped silicon product particles.

The method of fabricating a particulate product may comprise the furtherstep (c) of porosifying said sample of silicon and/or porosifying the orat least one of the silicon product particles formed from the sample ofsilicon.

If the porosification step (c) involves the porosification of the or atleast one of the silicon product particles, then the porosification maybe performed in such a manner that it does not substantially alter thesize and/or shape of the silicon product particle.

The method of fabricating a particulate product may comprise the furtherstep (d), performed prior to steps (b) and (c), of forming the sample ofsilicon by depositing a layer of polycrystalline silicon on a substrate.

The particle forming step (b) may be performed prior to or after step(c). In other words the silicon product particles may be formed fromporous or non-porous silicon.

Advantageously the particle forming step (b) is performed after step (c)and comprises the step of mechanically crushing said porous silicon.

Preferably the sample of silicon comprises a silicon wafer and step (b)comprises the step of etching the wafer. The step of etching the wafermay performed in such a manner that a multiplicity of monodispersedsilicon product particles are formed; said monodispersed silicon productparticles having a uniform size and/or shape.

The step (b) may comprise the step of photolithographically etching thewafer.

Advantageously step (b) is performed in such a manner that the or atleast one of the silicon product particles has a size in the range 1 nmto 500 μm. More advantageously the or at least one of the siliconproduct particles has a size in the range 1 nm to 250 μm.

Step (b) may be performed in such a manner that the or at least one ofthe silicon product particles has a size in the range 10 μm to 100 μm.Step (b) may be performed in such a manner that the or at least one ofthe silicon product particles has a size in the range 10 μm to 70 μm.Step (b) may be performed in such a manner that the or at least one ofthe silicon product particles has a size in the range 1 μm to 15 μm.

Step (c) may comprise the step of anodising said sample of silicon. Step(c) may comprise the step of electrochemical etching said sample ofsilicon.

Preferably the step (c) comprises the step of applying a stain etchsolution to the or at least one of the silicon product particles and/orapplying a stain etch solution to the sample of silicon. More preferablythe stain etch solution comprises hydrofluoric acid and an oxidisingagent. Yet more preferably the stain etch solution compriseshydrofluoric acid and one or more of: nitric acid, sodium nitrite, andchromium trioxide. Even more preferably the stain etch solutioncomprises hydrofluoric acid and nitric acid; wherein the concentrationof hydrofluoric acid, in the stain etch solution, is in the range 10 to30 mol per litre and the concentration of nitric acid, in the stain etchsolution, is in the range 0.0016 to 0.32 mol per litre.

Advantageously the process for fabricating a particulate productcomprises the step of bombarding the or at least one of the siliconproduct particles, and/or bombarding the sample of silicon, with one ormore of: ions, neutrons, and electrons; and further comprises the stepof porosifying the silicon contained in the or at least one of thesilicon product particles, and/or contained in the sample of silicon,that has been so bombarded.

Preferably the step of porosifying the silicon contained in the or atleast one of the silicon product particles, and/or the silicon containedin the sample of silicon, comprises the step of light assistedporosification.

Preferably step (d) comprises the step of reacting a silicon containinggas in the region of the substrate. More preferably step (d) comprisesthe step of pyrolysing a silane and/or halogen substituted silane in theregion of the substrate. Yet more preferably step (d) comprises the stepof pyrolising SiH₄ in the region of the substrate.

Preferably step (b) comprises the steps: (i) mechanically processing thesample of silicon in such a manner that at least one intermediatesilicon particle, is/are formed, the or each intermediate particlehaving a volume that is less than that of the sample of silicon fromwhich it was formed; and (ii) applying a size reduction etch to the orat least one of the intermediate silicon particles, the etch beingperformed in such a manner that it reduces the size of the or at leastone of the intermediate silicon particles.

The size reduction etch (ii) may be performed in such a manner that itdoes not substantially alter the shape of the or at least one of theintermediate silicon particles.

Preferably the step (i) of mechanically processing the sample of siliconcomprises the step of dicing and/or sawing and/or milling and/orcrushing and/or polishing and/or grinding the sample of silicon.

Advantageously the step (ii) of mechanically processing the sample ofsilicon is performed in such a manner that a multiplicity ofmonodispersed intermediate silicon particles are formed, eachmonodispersed intermediate silicon particle having substantially thesame size and/or shape.

The size reduction etch (ii) may comprise a wet etch. The size reductionetch (ii) may comprise an isotropic etch. The size reduction etch (ii)may comprise a planar etch.

Advantageously the step (ii) of applying a size reduction etch comprisesthe step of applying a size reduction etch solution to the or at leastone of the intermediate silicon particles, the etch solution comprisinghydrofluoric acid and nitric acid. More advantageously the sizereduction etch (ii) solution comprises hydrofluoric acid, nitric acid,and ethanoic acid, the concentration of the hydrofluoric acid, in thesize reduction etch solution, being in the range 1.1 to 7.7 mol perlitre, the concentration of nitric acid, in the size reduction etchsolution, being in the range 10.4 to 14.2 mol per litre, and theconcentration of ethanoic acid, in the size reduction etch solution,being in the range 0.0 to 1.74 mol litre.

According to a fifth aspect, the invention provides a method offabricating a carrier component, suitable for use in a microprojectiledelivery device, comprising the steps: (a) taking a sample of siliconand (b) forming particles from the silicon, step (b) being performed insuch a manner that a particulate product comprising at least onemicroprojectile is formed, and (e) assembling the particulate productwith a carrier body in such a manner that the product is retained by thebody to form a carrier component.

The silicon may comprise bulk crystalline silicon and/or polycrystallineand/or porous silicon.

The method of fabricating a carrier component may comprise the furtherstep (c) of porosifying said sample of silicon.

Step (c) may be performed before or after step (b).

The method of fabricating a carrier component may comprise the step (d),performed prior to steps (b) and (c), of forming the sample of siliconby depositing a layer of polycrystalline silicon on a substrate.

Advantageously step (b) is performed in such a manner that the or atleast one of the microprojectiles has a size in the range 1 nm to 500μm. More advantageously the or at least one of the microprojectiles hasa size in the range 1 nm to 250 μm.

Step (b) may be performed in such a manner that the or at least one ofthe microprojectiles has a size in the range 10 μm to 100 μm. Step (b)may be performed in such a manner that the or at least one of themicroprojectiles has a size in the range 10 μm to 70 μm. Step (b) may beperformed in such a manner that the or at least one of themicroprojectiles has a size in the range 1 μm to 15 μm.

Preferably step (d) comprises the step of reacting a silicon containinggas in the region of the substrate. More preferably step (d) comprisesthe step of pyrolysing a silane and/or halogen substituted silane in theregion of the substrate. Yet more preferably step (d) comprises the stepof pyrolising SiH₄ in the region of the substrate.

According to a sixth aspect, the invention provides a method offabricating a delivery device comprising the steps: (a) taking a sampleof silicon, and (b) forming particles from the silicon, step (b) beingperformed in such a manner that at least one microprojectile is formed,and (f) assembling an activatable gas source and the microprojectile(s)to form a microprojectile delivery device, the gas source andmicroprojectile(s) being arranged in such manner that, when activated,the gas source causes gas to impart kinetic energy to themicroprojectile(s).

The method of fabricating a microprojectile delivery device may comprisethe further step (c) of porosifying said sample of silicon.

The silicon may comprise bulk crystalline silicon and/or polycrystallineand/or porous silicon.

Step (b) may be performed before or after step (c).

The method of fabricating a microprojectile delivery device may comprisethe step (d), performed prior to steps (b) and (c), of depositing alayer of polycrystalline silicon on a substrate.

Preferably step (d) comprises the step of reacting a silicon containinggas in the region of the substrate. More preferably step (d) comprisesthe step of pyrolysing a silane and/or halogen substituted silane in theregion of the substrate. Yet more preferably step (d) comprises the stepof pyrolising SiH₄ in the region of the substrate.

Advantageously step (b) is performed in such a manner that the or atleast one of the microprojectiles has a size in the range 1 nm to 500μm. More advantageously the or at least one of the microprojectiles hasa size in the range 1 nm to 250 μm.

Step (b) may be performed in such a manner that the or at least one ofthe microprojectiles has a size in the range 10 μm to 100 μm. Step (b)may be performed in such a manner that the or at least one of themicroprojectiles has a size in the range 10 μm to 70 μm. Step (b) may beperformed in such a manner that the or at least one of themicroprojectiles has a size in the range 1 μm to 15 μm.

According to an seventh aspect the invention provides a method oftransfecting at least one cell, the method comprising the steps: (a)taking a microprojectile comprising silicon, (b) combining the particlewith a sample of DNA, and (c) implanting the microprojectile in the orat least one of said cells by microprojectile implantation.

Preferably the microprojectile comprises porous and/or polycrystallinesilicon.

According to an ninth aspect the invention provides the use of porousand/or polycrystalline silicon, in the preparation of a microprojectilefor the delivery of a physiologically active substance to a subject.

Whilst many countries do not, yet, permit the patenting of methods oftreatment of the human or animal body by surgery or therapy, there aresome (e.g. USA) who do. In order for there to be no doubt about theParis Convention priority entitlement to such an invention in thosecountries that do permit it, the invention also comprises the treatment,therapeutic or prophylactic, of a disorder of the human or animal bodyby microprojectile implantation of at least one microprojectilecomprising porous and/or polycrystalline silicon; and allowing therelease of an beneficial substance which helps to alleviate orameliorate the disorder, or to prevent the disorder from occurring.

A “beneficial substance” is something beneficial overall: it could be atoxin, toxic to undesirable cells or to interfere with an undesirablephysiological process. For example, anti-cancer substances would beconsidered “beneficial”, even though their aim is to kill cancer cells.

According to an eleventh aspect, the invention provides a use of aparticulate product comprising at least one silicon particle for themanufacture of a medicament for the treatment of a patient bymicroprojectile injection.

Advantageously the or at least one of the silcon particles is amicroprojectile.

Advantageously the or at least one of the silicon particles furthercomprises an active substance.

The active substance may comprise one or more of: a pharmaceuticalmaterial, a biological material, a genetic material, a radioactivematerial, an antibacterial agent, and luminescent agent.

The active substance may comprise one or more of: insulin, lidocaine,anaesthetic, alprostadil, calcitonin, DNA, RNA, peptide, cytokine,hormone, antibody, cytotoxic agent, adjuvant, steroid, and protein.

The active substance may comprise one or more of: GnRH, Goserilin,Leuprordin Acetate, Triptordin, Buserelin, a GnRH agonist, a GnRHsuperagonist, a GnRH antagonist, a GnRH homologue, a GnRH analogue, anda GnRH mimic.

For the purposes of this specification the term active substance meansany substance to be transferred into a target cell or tissue or into apatient.

Advantageously the active substance comprises DNA or RNA.

Advantageously the or at least one of the silicon particles comprisesporous silicon having a porosity between 1% and 90%. More advantageouslythe porous silicon has a porosity between 10% and 80%.

Preferably the or at least one of the silicon particles may have a sizein the range 100 nm to 500 μm. More preferably the or at least one ofthe silicon particles has a size in the range 100 nm to 250 μm.

The or at least one of the silicon particles may have a size in therange 10 μm to 100 μm. The or at least one of the silicon particles mayhave a size in the range 10 μm to 70 μm. The or at least one of thesilicon particles may have a size in the range 1 μm to 15 μm.

Advantageously the particulate product comprises at least fivesubstantially single sized silicon particles, each single sized siliconparticle having a volume that is substantially identical to the volumeof the other single sized particles. More advantageously the particulateproduct comprises at least ten substantially single sized siliconparticles, each single sized silicon particle having a volume that issubstantially identical to the volume of the other single sizedparticles. Yet more advantageously the particulate product comprises atleast twenty substantially single sized silicon particles, each singlesized silicon particle having a volume that is substantially identicalto the volume of the other single sized particles.

Preferably the particulate product comprises a multiplicity of singleshaped silicon particles, each single shaped silicon particle having thesubstantially same shape as the other single shaped silicon particles.More advantageously each single shaped silicon particle has thesubstantially the same volume as the other single shaped siliconparticles. Yet more advantageously each single shaped silicon particleis substantially symmetric.

The total mass of the single shaped silicon particles, from which theparticulate product is at least partly formed, may be greater than 10%of the total mass of the particulate product. The total mass of thesingle shaped silicon particles, from which the particulate product isat least partly formed, may be greater than 50% of the total mass of theparticulate product.

The particulate product may comprise a multiplicity of silicon particlesand at least some of said silicon particles may be monodispersed.

Advantageously the or at least one of the silicon particles issubstantially symmetric. More advantageously the particualte productcomprises a multiplicity of substantially symmetric silicon particles,each substantially symmetric silicon particle being substantiallysymmetric.

The or at least one of the silicon particles may be substantially cubic.The or at least one of the silicon particles may be substantiallyspherical.

For the purposes of this specification the term “symmetric”, when usedto describe an object, means that the object comprises at least oneplane of symmetry and/or at least one axis of symmetry.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described by way of example,with reference to the accompanying drawings, in which:

FIG. 1 shows a schematic diagram of a microprojectile delivery deviceaccording to the invention;

FIG. 2 contains a SEM images of a silicon cubes formed by dicing asilicon wafer;

FIG. 3 shows an SEM image of silicon cubes and spheres produced by acombination of mechanical processing and etching;

FIG. 4 shows a high magnification SEM image of silicon cube that hasbeen porosified by stain etching;

FIG. 5 shows an SEM image of a section of a porous silicon cube;

FIG. 6 shows SEM images of bulk crystalline and porous silicon spheres;

FIG. 7 illustrates a method of fabricating a particulate productaccording to the invention;

FIG. 8 shows a schematic diagram of a plurality of microprojectilescomprising an array of microneedles;

FIG. 9(a) shows a backscattered electron micrograph of porous siliconparticles embedded in a gelatine target;

FIG. 9(b) shows an EDX elemental distribution corresponding to the FIG.9(a) image for porous silicon particles embedded in gelatine;

FIG. 10 shows a Kα energy dispersive x-ray elemental distribution mapfor porous silicon particles embedded in a gelatine target;

FIG. 11 shows an SEM image of bulk crystalline silicon cube that hasbeen recovered from a gelatine target; and

FIG. 12 shows optical microsope image of a silicon particle trajectoryin gelatine block in cross-section.

DETAILED DESCRIPTION OF THE INVENTION

Microprojectile Delivery Device

FIG. 1 shows a schematic diagram of a microprojectile delivery device,generally indicated by 10, according to the invention. The deliverydevice 10 comprises a carrier component, generally indicated by 12, agas source 13 and exit system 15. The carrier component 12 comprises acarrier body 16 and a multiplicity of microprojectiles 17. An activesubstance will typically form part of the microprojectile 17. The activesubstance may be coated on of the surface of each microprojectile orlocated, at least partly, in interior of each microprojectile. Thefabrication of microprojectiles 17 and the association of an activesubstance with the microprojectiles 17 is discussed below.

The carrier body 16 is tubular in shape and the microprojectiles 17 areadhered to the interior surface of the carrier body 16. The carriercomponent 12 is connected, via the valve 13 b, to the gas source 13. Thegas source 13 comprises a gas cylinder 13 a and a gas valve 13 b. Thegas cylinder 13 a, containing a compressed gas, is activated by openingthe valve 13 b for a short interval. Activation of the gas source 13 inthis way causes a pulse of gas to be released. The released gas passesthrough the carrier component 12 and as it does so some of themicroprojectiles 17 are dislodged and entrained in the flow of gas. Themicroprojectiles 17 pass through the exit system 15 and travel to atarget 18. The target 18 may comprise cells or tissue. For example thetarget 18 may be a human or animal patient. The design of the exitsystem 15 will determine the manner in which the microprojectiles 17 aredelivered to the target 18; for example it may determine the trajectoryor the degree of dispersion of the microprojectiles 17.

There are a number of methods by which a particulate product comprisingsilicon may be fabricated.

Fabrication of a Particulate Product Comprising Silicon Particles

The following processes (numbered 1 to 12) each describe the fabricationof a particulate product comprising at least one silicon particle. Eachof these processes may be used to fabricate a particulate productcomprising at least one silicon microprojectile.

Process 1

A silicon wafer may be anodised in an HF solution, for example a 50%aqueous or ethanolic solution, to form a layer of porous silicon. Theanodisation may be carried out in an electrochemical cell by standardmethods such as that described in U.S. Pat. No. 5,348,618. For example awafer may be exposed to an anodisation current density of between 5 and500 mAcm² for between 1 and 50 minutes. In this way a layer of poroussilicon having a porosities in the range 1% to 90% may be fabricated.The porous silicon layer may then be detached from the underlying bulksubstrate by applying a sufficiently high current density in arelatively dilute electrolyte, for example a current density of greaterthan 50 mAcm⁻² for a period of 10 seconds. Alternatively the anodisedwafer may be treated ultrasonically to detach the layer of poroussilicon and to break up the layer into particles of porous silicon.Exposure to ultrasound in this way may be performed in a solvent, thesolvent being chosen to minimise agglomeration of the resultingparticles. Some control over particle sizes may be achieved bycentrifuging the resulting suspension to separate the different particlesizes. The porous silicon particles may also be sized by allowing thesuspension gradually settle as described in Phys. Solid State 36(8)1294-1297 (1994). Control over silicon particle shape may be attained bysize and shape distribution analysis such as laser diffraction analysis,electrozone sensing, hydrozone focussing, and sheath flow technology.

Silicon powders of micron particle size are available commercially. Suchcommercially available particulate products have silicon particles thathave irregular shapes, and that exhibit a wide range of particle sizes.Nanometre size particles can be fabricated from silicon wafers byprocesses such as ball milling, sputtering, and laser ablation of bulksilicon.

Process 2

A silicon on insulator (SOI) wafer may be photolithographically etchedby standard wet etch or dry etch techniques such as those described inPCT/GB99/02381. The etch may be performed in such a manner that an arrayof silicon microprojectiles are formed on the oxide substrate. Themicroprojectiles may have dimensions in the range 10 to 250 μm. Themicroprojectiles can be detached from the oxide substrate by standard HFsoak. The microprojectiles can then filtered off, washed and dried priorto porosification. In this way a particulate product comprising poroussilicon particles of monodispersed size and shape may be obtained.

Alternatively, and more specifically, a 20 to 30 Ωcm p type (100)silicon wafer with a 10 micron thick p++ top layer is coated on bothsides with 100 nm of silicon oxide. The silica layer on the back of thewafer is then patterned with a membrane photomask and reactive ionetched to define the wafer area to be thinned. A supported 10 micronthick membrane is then realised by wet etching through from the back ofthe wafer to the p++/p− interface. For a 475 micron thick wafer and KOHat 80C. this takes 10 to 15 hours. Thick photoresist is then depositedin the back etched cavity as a support for the membrane and as asubstrate from which the silicon particles may be removed. Positivephotoresist is spun on the front face of the wafer and pattered with aphotomask containing thousands of 10×10 micron spaced squares. Thesilica and p++ membrane are then reactive ion etched. The thickphotoresist/diced silicon membrane is then removed from the wafer andplaced in a centrifuge tube. The silicon cubes can then be released bydissolving the photoresist in acetone, and collected by centrifugation.

Porosification of the above silicon particles may be achieved bystandard stain etching as described in J Applied Physics 78(6)p4273-4275 (1995), or light-assisted stain etching as described inPhysical Chemistry Chemical Physics 2(2):277-281, 2000. Thelithographically based approach allows the fabrication of siliconparticles having a well defined shape and narrow size distribution.

Process 3

A multiplicity of silicon cubes may be fabricated by dicing a siliconwafer, using MicroAce 3 automated dicing equipment incorporating a 75micron wide blade impregnated with 4 to 6 micron diamond powder. Byappropriately programming of the MicroAce, cubes having substantiallythe same size as each other are formed, as shown in FIGS. 2 a to 2 c.The cube dimensions are approximately equal to the thickness of thewafer from which they were formed. Cubes having dimensions in the range100 microns to 2 mm may be fabricated by this method. The cubes shown inFIGS. 2 a and 2 b are substantially monodispersed, having uniformdimensions and shapes. As can be seen the cube shown in FIG. 2 c issubstantially symmetric, though there is a small amount of saw, damagein the form of small chips and irregularities on four of the six faces.

Process 4

Further mechanical processing may then performed on the cubes, formed byprocess 3, to convert them into substantially spherical beads. The cubeswere introduced to a spherical drum, lined with an abrasive paper. Thedrum comprises an array of directed nozzles, located at the centre ofthe drum, through which a stream of compressed gas flows. The compressedgas causes the silicon particles to be blown around a circulartrajectory within the drum. Experiments were performed, on 2 mm widecubes, at 20 to 40 psi for 5 to 60 minutes using a 200 micron filter toremove unwanted silicon dust. Mechanical processing in this way yieldedsubstantially spherical silicon beads having a diameters approximatelyin the range 1.6 mm to 1.2 mm. The method used to fabricate theseparticles is similar to that disclosed in “The Review of ScientificInstruments Vol 36(7) p957 to 958 (1965).

Process 5

The silicon cubes, fabricated by process 3, may also be converted tosilicon spheres by particle milling. This involves tumbling the siliconparticles in an abrasive medium. Suitable particulate milling mediainclude industrial diamond powder, ceramic micro particles, andstainless steel or zirconia balls.

Process 6

Alternatively the cubes, fabricated by process 3, may be converted tosilicon spheres by grinding the silicon particles between two rotatingplates of sufficiently hard material such as tungsten carbide orpreferably plates that have been covered by a thin film of hardabrasive. For example, monodspered silicon spheres may be fabricated,from cubes generated by process 3, by polishing between two flat glassplates covered by 600 grit wet and dry paper. Silicon cubes are firstplaced onto the centre of the lower plate and then covered in a thinlayer of oil, such as Hyprez fluid. The second plate is then placed ontop of the cubes and moved in a circular motion. Once the edges of thecubes have been removed, so that the the silicon particles act asbearing for the two plates, a mass of several kg may be applied to theupper plate. Grinding between the two glass plates may be for a periodranging from a few minutes to a several hours.

FIG. 6(a) shows a multiplicity of silicon spheres that have beenfabricated by the process 6 method.

Process 7

Silicon particles fabricated by one or more of processes 3 to 6 mayfurther be subjected to a size reduction etch that reduces the size ofthe particles and also reduces surface damage resulting from mechanicalprocessing. The size reduction etch solution may be formed by combining5 volumes of 70% aqueous nitric acid, 1 volume a 40% aqueoushydrofluoric acid, and 1 volume substantially pure ethanoic acid; thissolution will be referred to as “5:1:1 etch solution”.

Silicon particles, fabricated by one or more of processes 3 to 6, wereetched in the presence of silicon discs, each disc having a diameter of1 cm, the mass of silicon discs required was 0.8 g per 35 ml of the5:1:1 etch solution. FIG. 3 shows four silicon cubes and six siliconspheres of varying sizes. The cubes shown in FIG. 3 were obtained byexposing cubes fabricated by process 3 to the 5:1:1 solution for periodsof between 5 and 60 minutes minutes, the length of each side varies from2 mm (for the largest cube) to 380 microns (for the smallest cube). Thespheres shown in FIG. 3 were obtained by exposing spheres fabricated byprocess 4 to the 5:1:1 etch solution for periods between 5 and 30minutes, the sphere diameters vary from 1.1 mm (for the largest sphere)to 350 microns (for the smallest sphere).

Process 8

Particulate products, fabricated by one or more of processes 3 to 7, orby the photolithographic technique forming part of process 2, may beporosified by stain etching the silicon particles.

A stain etch solution comprising hydrofluoric acid and nitric acid wasemployed. The stain etch solution was formed by combining 100 volumes of40% aqueous hydrofluoric acid solution with 1 volume of 70% aqueousnitric acid solution; this stain etch solution will be referred to asthe “100:1 solution”. The 100:1 solution may be applied to theparticulate product for a period of five minutes to yield siliconparticles having a 3.9 micron, 47% porosity, layer of porous silicon.FIG. 4 shows an SEM image of a cube, having sides of length 100 microns,fabricated by process 3, and porosified by treatment with the 100:1 etchsolution for a period of 10 mintutes. FIG. 5 shows an SEM image of asection of a silicon cube, having sides of length 100 microns,fabricated by process 3, and porosified by treatment with the 100:1 etchsolution for a period of 10 minutes. The section shown in FIG. 5 is acorner of the cube, and the image shows a layer of porous silicon thathas been formed at the periphery of the cube.

The FIG. 4 image may be compared with that of FIG. 2 c. The FIG. 2 cimage was taken at the same maginifcation as the FIG. 4 image, and showsa cube having the same dimensions as the FIG. 4 cube. However, the FIG.2 c cube has not been porosified. A comparison of the two images showsthat porosification using the 100:1 etch solution need not result in anysubstantial change in size or shape. This is markedly illustrated by thecontinued presence of saw damage in the FIG. 4 cube.

FIGS. 2 a and 2 b show monodispersed un-porosified silicon particles,and FIGS. 2 c and 4 show that porosification causes negligible change tosize and shape. Therefore the results shown in FIGS. 2 and 4 show thatit is possible to fabricate a monodispersed particulate productcomprising porous silicon particles having a largest dimension less than500 microns.

The use of a stain etch may not only cause porosification of the sampleof silicon to which it is applied, but it may also dissolve at leastsome of the porous silicon that is so formed. This dissolution may limitthe thickness of porous silicon that can be achieved by stain etching.

FIG. 6(b) shows four porous silicon spheres that have been fabricated bythe process 6 method followed by stain etching according to the process8 method. FIG. 6(b) shows a higher magnification image of one of theporous silicon spheres shown in FIG. 6(b).

Process 9

Ion bombardment of the particulate product may, at least partially,solve the problem of dissolution associated with stain etching. Forexample Si, F, Cl, H, He, and Ar ions, may be used to bombard thesilicon of the particualte product. Alternatively neutrons or electronsmay also be used. Such bombardment introduces point defects or extendeddefects in the sample of silicon. The presence of the defects allows theuse of a less chemically aggressive stain etch solutions that, for agiven rate of porosification, cause less dissolution. The use ofparticle bombardment followed by porosification in this way is describedin Jpn J Appi Phys Vol 31 (5A) p L560-L563 (1992) and in Semiconductors30 (6) p 580-584 (1996). The use of lighter elements may be ofparticular value, for example 2 MeV H+ has a projected range of 50microns, potentially opening the way for the use of less aggressivestain etch solutions over a range of several tens of microns.

Process 10

Silicon particles may also be fabricated using polycrystalline silicon,by the process steps illustrated in FIG. 7. A layer of phosphosilicateglass 21 (PSG) may be deposited on a silicon substrate 22. Thedeposition may be performed using atmospheric pressure CVD by reactingpure silane and phosphine with oxygen in a nitrogen stream. The PSG 21may then be patterned by conventional techniques to form an array ofbase structures 23. A layer of polycrystalline silicon (not shown inFIG. 7) can then deposited by pyrolysis of silane using low pressureCVD. The polycrystalline silicon layer is then patterned, by standardetching techniques, in such a manner that each base structure isenveloped in an island layer of polycrystalline silicon 24, and that theisland layer is also bonded to the silicon substrate adjacent to thebase structure. Heating the polysilicon layer to temperatures between950 and 1100C for 10 to 30 minutes causes the polysilicon layer todeform (as shown in FIG. 7 d) as a result of the release of P₂O₅ fromthe PSG. By selecting the correct form of patterning and conditions thedetached silicon particles comprising shell like structures may be usedfor microprojectile implantation.

Process 11

Silicon has a low density relative to some materials (such as gold andtungsten) currently used in the fabrication of microprojectiles forintercellular delivery. The ability of a microprojectile to penetrate acell wall or tissue depends to some extent on its momentum and hence onits mass. It may therefore be necessary, for some applications, toincrease, the density of a silicon microprojectile. This may be done byintroducing an element or compound to the microprojectile that has adensity greater than that of silicon. There are a number of ways inwhich such elements may be introduced. A review of porous siliconimpregnation is presented in the book “Properties of Porous Silicon”,Chapter 1.9, p 66 to 76, Published by INSPEC (ISBN 085296 932 5). Afurther group of methods of impregnation is described in PCT/GB99/01185.For example silver nitrate powder may be placed onto the surface of asample of porous silicon. The silver nitrate and porous silicon may thenbe heated in an argon atmosphere until the nitrate is observed to meltand decompose. The molten nitrate enters the pores where it decomposesthereby depositing silver within the porous silicon.

Process 12

As mentioned in the last paragraph the problem associated with silicon'slow density may be overcome by impregnation of the microprojectile withan element or compound. A further way in which this problem can beovercome is to form the microprojectiles from an array of microneedlesas shown in FIG. 8. Such an array 31 may be formed by standard wetetching techniques such as those described in IEEE Transactions inBiomedical Engineering Vol 38, No 8, August 1991, p 758 to 768.

Specifically, two sets of deep (200 micron) orthogonal cuts are madeinto a 380 micron thick wafer. The wafer is rotated by 90 degreesbetween the first set of n cuts in one direction, and the second set ofm cuts in the orthoganal direction. This sawing does not cut through thewafer at any point but creates an array of n×m square columns having anaspect ratio determined by the spacing of the cuts. For a 75 micron wideblade and a pitch of 175 microns one creates 100 micron wide squarecolumns. The subsequent etching processes to define dart-like shapesthen have 3 steps. The first chemical etch is to remove saw damage,isotropically reduce the width of the columns and round the edges at thebase of the columns. It utilizes an HF: HNO3 etch (eg 5% to 95%) that isconducted with vigorous agitation. The second chemical etch is performedunder static conditions that promote preferential attack of the top ofthe columns to create pointed tips and a tapered shaft. The third etchstep is to create a mechanical weakness at the base of the columns whichfacilitates their detachment from the underlying silicon membrane. Thiscan be achieved by the use of dry etch conditions that undercut thecolumns.

FIG. 8 shows a schematic diagram of an array of silicon microneedles 31that is integral with a substrate 32. The substrate 32 may form aparticle carrier or part of a particle carrier suitable for loading in amicroprojectile delivery device similar to that shown in FIG. 1. Thesubstrate 32 and gas source may be arranged such that the gas impactswith the side of the substrate 32 opposite to that on which themicroneedles 31 are formed. This differs from the arrangement shown inFIG. 1 in which the surface in which the microprojectiles are arrangedis roughly parallel to the direction of gas flow. Impact of the gas withthe substrate 32 would then cause the microneedles 31 to break away fromthe substrate and to become incident upon the target. The process may befacilitated by forming a narrow neck portion 33 adjacent to thesubstrate.

The narrow neck portions at the base of the needles may be formed bystandard deep dry etching techniques in combination with an etch stop(eg silicon oxide).

Incorporation of an Active Substance into a Particulate Product

There are a number of methods by which the particulate product may bemade to comprise the active substance. This section describes thefabrication of silicon particles comprising an active substance. Themethods of fabrication described in this section may be suitable forfabricating silicon microprojectiles comprising an active substance.

The method selected will depend on a number of criteria including: (a)the nature of the active substance to be loaded in the silicon particle,(b) the dose or loading required, (c) the pharmacokinetic releaseprofile required for optimal delivery of the active substance inquestion, (d) whether a derivatised form of silicon is preferred, (e)the hydrophilicity/hydrophobicity profile of the active substance to beloaded, and (f) whether an active substance release mechanism isrequired over and above the rate of dissolution of the silicon particlein order to effect drug release.

The following methods may be applied to load porous siliconmicroprojectile implants:

Liquid or Solution Phase Loading

The active substance may be converted to a liquid form by dissolution orsuspension in an aqueous, organic or amphypathic phase. Alternativelythe active substance may be a liquid at room temperature or it may bemade liquid by exposure to an appropriate temperature and/or pressure.The active substance, in liquid form, can then be taken up by poroussilicon particles by bringing the particles into direct contact with theactive substance. Porous silicon exhibits substantial capilliarity andas a consequence, the liquid phase active substance may be drawn intothe porous silicon material by capillary action. If a solution orsuspension of the active substance has been used, the microprojectilemay then be dried by conventional freeze drying or other routinelypracticed drying techniques. The steps of liquid/solution loadingfollowed by drying may be repeated a number of times to increase theamount of active substance in each porous silicon particle.

Alternatively the silicon particles comprising porous silicon and anactive substance may be fabricated by forming a disc of porous siliconin the manner described in process 1. Such a disc may then be used as afilter for a suspension or solution of an active substance in a suitableliquid carrier or solution. As the solution or suspension passes throughthe porous silicon filter, the active substance may be deposited ontothe porous silicon. The porous silicon disc, on which the activesubstance has been deposited, may then be converted to a particulateproduct, comprising silicon particles, by the method described inProcess 1.

Solid Phase Loading

Finely divided porous silicon may be combined with a finely divided formof an active substance in the solid phase by such techniques as spraycoating techniques and pressure based techniques. The finely dividedsilicon/active substance is moulded to form silicon particles of therequired mass, shape and dimensions. The use of highly porous siliconcomprising quantum wires is of particular value for this technique, thefinely divided silicon being formed by crushing the porous silicon.

Derivatisation and Sequestration

Porous silicon may be derivatised by techniques similar to thosedescribed in J M Buriak, J Chem Soc, Chem Commun p 1051, 1999, in such amanner that a biomolecule, having a high affinity for a particularactive substance, is bonded to the surface of the porous silicon.Biomolecules that might be suitable for this application includeantibodies, enzymes, hormones, receptors, proteins, and peptides. Thederivatised porous silicon may then be combined with the activesubstance by methods described in this section, the active substanceforming a bond to the porous silicon by means of the biomolecule. Theporous silicon may then be converted to a particulate product by themethod described in Process 1.

Electronic Precipitation

A further method by Which porous silicon may be combined with an activesubstance is by electronic precipitation. A sample of porous silicon maybe placed in a solution containing cations or anions of the activesubstance. The porous silicon may then be biased in such a manner thatthe cations or anions of the active substance are attracted to theporous silicon. The porous silicon may then be converted to aparticulate product by the method described in Process 1.

The active substance (for example DNA) may be dissolved or suspended ina suitable solvent, the microprojectiles may then be incubated in theresulting solution for a period of time. The active substance may thenbe deposited on the surface of the microprojectiles. If themicroprojectiles comprise porous silicon, then a solution of the activesubstance may be introduced into the pores of the porous silicon bycapillary action. Similarly if the microprojectiles have a cavity thenthe solution may also be introduced into the cavity by capillary action.If the active substance is a solid but has a sufficiently high vapourpressure at 20C. then it may be sublimed onto the surface of themicroprojectiles. If a solution or suspension of the active substancecan be formed then the substance may be applied by successive immersionin the solution/suspension followed by freeze drying.

Microprojectile Injection of Bulk Crystalline and Porous SiliconParticles into Tissue Simulant.

Two types of particulate product were tested. The first type of productcomprises porous silicon microprojectiles produced by process 1, and thesecond particulate product comprises cubic silicon microprojectilesfabricated by process 3. The density of the porous silicon particles wasapproximately 1.1 g cm⁻³. The first and second particulate products wereaccelerated, from an initial stationary state, towards a target tissuesimulant.

The target tissue simulant comprised gelatine, and was fabricatedfollowing a modified NATO standard procedure AC/225114 (1980). Drygelatin powder was mixed with water at a concentration of 20% by weight.The mixture is gelatinous and opaque. Then, without stirring, it washeated to 50C. to yield a clear, easy flowing liquid. Any foam orbubbles on the surface were skimmed off, prior to pouring the liquidinto suitable plastic moulds. The mixture was gradually cooled while inthe mould to a temperature of 20C. and then stored at 10C. for two daysprior to use.

The two types of particulate product were accelerated using a 0.5 inchdiameter Browning slave barrel device and 3N Vihtavouri propellant.During acceleration along the barrel, the particulate product was housedin a cylindrical cavity with a nylon sabot. This housing was selectivelystopped from reaching the gelatine block by a stainless steel “stripperplate” containing a hole that allows the passage of the particulateproduct, but not the sabot. The speed of sabot and its contents, uponexiting the barrel was 700 metres per second. The speed was measuredusing a Terma Electronik AS Doppler Instrument (DR5000 Model), triggeredby the muzzle flash.

FIG. 12 shows a typical cross sectional image, from an opticalmicroscope, showing a cavity formed in the gelatine as a result of themicroprojectile injection of the silicon microprojectiles.

FIG. 9(b) shows an image taken from the region of the cavity close tothe point at which the microprojectiles enter the gelatine. The FIG.9(b) image is an electron micrograph of the gelatine in this region, andit shows porous silicon particles (pale grey) surrounded by the cavity(dark grey). The corresponding EDX elemental distribution is shown inFIG. 9(b). FIG. 10 shows a Kα energy dispersive x-ray elementaldistribution map for porous silicon particles embedded in the sameregion. FIGS. 9 and 10 show that wholly porous silicon microprojectileshave acquired sufficient momentum to penetrate the tissue simulant. FIG.11 shows an SEM image of a cube that has been retrieved from the tissuesimulant. The cube had penetrated several mm into the gelatine. Theimage shows that the cube has been substantially undamaged by theacceleration and deceleration caused by the microprojectile injection.

1. A particulate product comprising monodispersed porous siliconparticles, at least one of the porous silicon particles comprisingporous silicon obtainable from a sample of silicon by one or more of:stain etching, anodization, and electrochemical etching.
 2. Aparticulate product according to claim 1 wherein at least one of thesilicon particles comprises a cavity that is bounded, at least partly,by the silicon.
 3. A particulate product according to claim 1 whereineach of the monodispersed silicon particles has a dimension between 100nm and 500 microns.
 4. A particulate product according to claim 1wherein each of the monodispersed silicon particles has a largestdimension less than 500 microns.
 5. A particulate product according toclaim 1 wherein at least one of the silicon particles is amicroprojectile.
 6. A particulate product according to claim 1 whereineach of the monodispersed silicon particles is a microprojectile and atleast one of the microprojectiles comprises a high density materialhaving a density greater than that of bulk crystalline silicon.
 7. Aparticulate product according to claim 6 wherein the high densitymaterial is selected from one or more of: gold, tungsten, platinum,iron, nickel, molybdenum, silver, palladium, erbium, iridium, rhenium,and cobalt.
 8. A particulate product according to claim 1 or 4 whereinat each of the monodispersed silicon particles is symmetric.
 9. Aparticulate product according to claim 8 wherein at least one of thesymmetric silicon particles is spherical.
 10. A particulate productaccording to claim 8 wherein at least one of the symmetric siliconparticles is cubic.